Juan, PI has a very broad fluorescence range, and fluoresces well into the 675nm region (see the attached PI ex/em spectrum, borrowed from Molecular Probes). There's no surprise, then, that you'll see PI signal into "FL3" . . . in fact, you can use this. By shifting your PI detection to 675nm (FL3), you can a) improve the resolution between PI and FITC, and b) you can then combine PI with PE nicely (usually you'll have to reduce the PI concentration . . .) This question again illustrates a problem that I've pointed out before concerning a disconnect between a user's awareness of what these FL# designations are, and the actual wavelengths they detect. I've stated before that we should be aware that these channels are arbitrary, and only defined as specific by the filters we've selected. So, FL1 is FITC because we use a 530/30 bandpass; FL2 is PE because we choose a 575/25 bandpass, but we can run PI there because "PI has a very broad fluorescence range," FL3 is PerCP . . . 675 nm bandpass . . . and on from there. It's certainly an advantage having worked with research instrumentation, where any channel is available for any signal. It gives one a perspective that's often lost when we consider closed-bench systems like the Calibur or the XL. But, again, a user of flow (especially an operator) should be aware of what these FL# designations are, and that we decided what these channels actually detect by choice of filter. Juan . . . I'm wouldn't be so concerned that you don't know . . . it is troubling that you say "Nobody in our facility can explain it . . " MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> "Juan Oliver M.D." <jao7@columbia.edu> 03/14/01 07:22PM >>> I have an embryonic pluripotent cell line that has a subpopulation of cells with a spontaneous strong signal with a FACScalibur in FL3 (7AAD); the remarkable finding is that this population of cells increases its signal one log in FL3 after PI addition (FL2); 10 micoliters of 0.1 mg/ml into ~ 1ml . Nobody in our facility can explain it; any suggestions would be appreciated. Thanks, Juan Oliver
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:10 EST